Examining the impact of laboratory housing temperature on murine CD28 and the response to anti-PD-1

项目摘要/摘要 几十年来，动物在生物医学研究中取得了重大的科学和医学突破， 产生基本的临床前数据，最终支持治疗的发现和开发 包括癌症在内的人类疾病。然而，当我们依靠动物模型来研究 癌症和癌症治疗的复杂性，这些临床前研究的成功率低得惊人， 可重复性，甚至更低的临床前到临床的成功率。根据护理和使用指南， 实验动物第8版，研究机构已标准化，最低限度的指导方针的住房， 饲养，和全面照顾实验动物，他们必须坚持。温和凉爽的环境 温度是动物圈养的一个关键方面， 研究啮齿动物的变化，由交感神经系统的激活和β- 肾上腺素能受体（β-AR）信号传导作为去甲肾上腺素的全身释放的结果。这是由于 补偿反应，称为非颤抖产热，由啮齿动物饲养在 低于其热中性区的温度（这是环境温度的范围， 基础代谢产生的热量足以维持体内平衡的核心温度。我们实验室有 先前确定的标准（ST），亚热中性实验室外壳温度导致 与小鼠相比，小鼠CD 8 + T细胞依赖性抗肿瘤免疫应答显著受损 置于热中性温度（TT）下。此外，我们已经证明，免疫检查点抑制剂 αPD-1是一种免疫疗法，最近已成功地作为治疗癌症的一线方法， 黑色素瘤，在治疗以β-AR依赖性方式圈养在TT的荷瘤小鼠中具有改善的功效。 虽然已发表的和初步的数据表明，在减少的抗肿瘤作用中，共受体CD 28的作用， 由于β-AR信号的增加，CD 8 + T细胞的功能，我们对CD 8 + T细胞的理解存在差距。 ST小鼠中CD 8 + T细胞活化和效应子功能降低的潜在机制。 因此，我们建议使用基因工程小鼠模型来精确地询问CD 28信号传导， 并检验标准住房温度会损害CD 8 + T细胞抗肿瘤免疫力， αPD-1通过受损的CD 28共刺激的体内疗效。我们将使用体外和体内方法， 研究了室温对CD 8 + T细胞CD 28表达和信号传导以及肿瘤的影响， 用αPD-1治疗的小鼠的浸润淋巴细胞。本提案中概述的研究包括： 确定先前未定义的机制的可能性，通过该机制，低温中性实验室动物圈养 温度影响癌症和免疫治疗模型的实验结果， 描述了一个存在于我们的动物模型中的被广泛低估的变量。

Project Abstract/Summary For decades, animals in biomedical research have yielded significant scientific and medical breakthroughs by generating the essential preclinical data that ultimately support the discovery and development of treatments for human diseases, including cancer. However, while we to rely on animal models to investigate the complexity of cancer and cancer therapies, these preclinical studies have alarmingly low success in reproducibility, and even lower preclinical-to-clinical success rates. As per the Guide for the Care and Use of Laboratory Animals 8th Edition, research institutions have standardized, minimum guidelines for the housing, husbandry, and overall care for laboratory animals that they must adhere to. A mildly cool ambient temperature is a critical aspect of animal housing that has been shown to elicit significant physiological changes to research rodents, driven by the activation of the sympathetic nervous system and increased β- adrenergic receptor (β-AR) signaling as a result of the systemic release of norepinephrine. This is due to the compensatory response, known as non-shivering thermogenesis, employed by rodents housed at temperatures that fall below their thermoneutral zone (which is the range of ambient temperatures at which heat generated by basal metabolism is sufficient for maintaining homeostatic core temperature. ) Our lab has previously established that standard (ST), subthermoneutral laboratory housing temperatures result in significant impairment to the murine CD8+ T cell-dependent anti-tumor immune response compared to mice house at thermoneutral temperatures (TT). Additionally, we have shown that the immune checkpoint inhibitor αPD-1, an immunotherapy that has recently seen success as a front-line approach to treating cancers like melanoma, has improved efficacy in treating tumor-bearing mice housed at TT in a β-AR-dependent manor. Although published and preliminary data indicate a role for the co-receptor, CD28, in the diminished anti-tumor function of CD8+ T cells as a result of increased β-AR signaling, a gap exists in our understanding of the mechanisms underlying the reduced CD8+ T cell activation and effector function in mice housed at ST. Therefore, we propose using genetically engineered mouse models to precisely interrogate CD28 signaling and test hypothesis that standard housing temperatures impairs CD8+ T cell anti-tumor immunity and the in vivo efficacy of αPD-1 via impaired CD28 co-stimulation. We will use in vitro and in vivo approaches to examine the effects of housing temperature on CD8+ T cell CD28 expression and signaling, as well as tumor- infiltrating lymphocytes in mice treated with αPD-1 therapy. The studies outline in this proposal have the potential to identify a previously undefined mechanism by which subthermoneutral laboratory animal housing temperatures influence experimental outcomes of cancer and immunotherapy models, while also characterizing a widely underappreciated variable that exists in our animal models.